原核的

Based on the cloning FeSOD gene of the HZNH, the recombinant prokaryotic expression vector, PQESOa/FeSOD, was constructed and digested with restriction endonuclease BamH I and Pst I to check its construction. The PQE30a/FeSOD was then transformed into E.coli Ml5 and induced with IPTG. The high expression in vitro was obtained and analyzed on SDS-PAGE gel. The*results showed that the target proteins held a 37% portion in whole bacterial proteins and consisted of two parts, the soluble proteins and inclusion bodies. The soluble proteins in the aqueous layer, checked by means of activities of FeSOD enzymes and analyzed by means of activities of isozymogram from PAGE, demonstrated the induced expression proteins had the active nature of FeSOD enzymes.

以克隆的特异种质烟草HZNH的FeSOD基因为基础，构建了原核表达载体PQE30a／FeSOD，经限制性内切酶BamH Ⅰ、Pst Ⅰ双酶切鉴定后，再转化入大肠杆菌M15中，通过IPTG诱导，得到高效体外表达，经SDS-聚丙烯酰胺凝胶电泳检测，表达的目的蛋白占总菌体蛋白的37％，可溶性和包涵体两种形式均有存在，上清中的可溶性蛋白经FeSOD酶活测定和同工酶活性谱带分析，表明诱导表达的上清中的目的蛋白为有活性的FeSOD酶。

Src l was an acidic cytolysin, which was reported forthe first time. The cDNA encoding the mature Src l was cloned into non-fusion expression vectorpBV220 and expressed successfully in E.coli DH5α in inclusion body. After washing anddenaturing-renaturing, the recombinant Src l protein was purified by Q Sepharose FastFlow ion exchange chromatograph and Phenyl Sepharose hydrophobic interactionchromatograph.

首先构建了Src I基因的非融合蛋白表达质粒pBV220-Src I，并成功地在原核细胞E.coli进行了重组表达，筛选出稳定表达的工程菌株DH5α，摸索了稳定表达的条件，摸索了包涵体的洗涤、变性和复性条件，摸索出了复性蛋白Src I的纯化工艺，纯化过程简单，经QSepharose Fast Flow阴离子交换层析和Phenyl Sepharose疏水层析两步纯化，就可获得了高纯度的重组蛋白样品(纯度达99％以上)，该纯化方法适合重组蛋白的大量纯化，为Src I的性质、结构和功能研究奠定了良好的基础。

Transgenic tobacco plants were obtained through screening with kanamycin. The transgenic tobacco plants could delay TMV infection for about 25 days compared with non-transgenic tobacco plants. Pokeweed antiviral protein Ⅱ is expressed with high level in summer leaves. The expression of PAPⅡ is regulated by season. The total RNA was extracted from pokeweed (Phytolacca americana L.) leaves in summer using the method of TRIzol and used as template to amplify the PAPⅡ gene by RT-PCR and then the gene was cloned into E. coli expression vector and secreted expression pPIC9K vector. The two vectors with PAPⅡ gene were then transferred into E. coli strain BL21 (DE3)-plysS and Pachia pastoris GS115 strain respectively. The specific protein was produced induced by IPTG and methanol.

由于美洲商陆抗病毒蛋白Ⅱ是一个受季节调控表达的蛋白，本实验以美洲商陆夏季叶片为材料，通过对其总RNA的提取、反转录、并用PAPⅡ的特异引物进行PCR扩增，对PAPⅡ进行了基因克隆、序列分析，结果扩增出来的PAPⅡ基因与已经报道的序列同源性是99.9％，然后将该基因构建到原核、真核表达载体上，分别转化了大肠杆菌和毕赤酵母并对它们进行了诱导表达，在两个表达系统中均获得了有活性的PAPⅡ表达蛋白，体外生物测定表明表达的蛋白均具有抑制病毒的活性，PAPⅡ基因在酵母中还没有表达的报道，这为获得具活性PAPⅡ蛋白提供了一种简便可行的方法。

The transgenic tobacco plants could delay TMV infection for about 25 days compared with non-transgenic tobacco plants.Pokeweed antiviral protein II is expressed with high level in summer leaves. The expression of PAPII is regulated by season. The total RNA was extracted from pokeweed (Phytolacca americana L.) leaves in summer using the method of TRIzol and used as template to amplify the PAPII gene by RT-PCR and then the gene was cloned into E.coli expression vector and secreted expression pPIC9K vector. The two vectors with PAPII gene were then transferred into E.coli strain BL21 (DE3)-plysS and Pachia pastor is GS115 strain respectively. The specific protein was produced induced by IPTG and methanol.

由于美洲商陆抗病毒蛋白Ⅱ是一个受季节调控表达的蛋白，本实验以美洲商陆夏季叶片为材料，通过对其总RNA的提取、反转录、并用PAPⅡ的特异引物进行PCR扩增，对PAPⅡ进行了基因克隆、序列分析，结果扩增出来的PAPⅡ基因与已经报道的序列同源性是99.9％，然后将该基因构建到原核、真核表达载体上，分别转化了大肠杆菌和毕赤酵母并对它们进行了诱导表达，在两个表达系统中均获得了有活性的PAPⅡ表达蛋白，体外生物测定表明表达的蛋白均具有抑制病毒的活性，PAPⅡ基因在酵母中还没有表达的报道，这为获得具活性PAPⅡ蛋白提供了一种简便可行的方法。

After thecomplete genome extraction of the strain was performed, the genomic DNA was partiallydigested by restriction enzyme Sau3AⅠ, the DNA fragments from 1 to 5Kb was clonedinto prokaryote expression vector pET-28a-c, and transformed host bacteria. The resultsshowed that we succeeded in constructing the gene expression library of haemophilusparasuis serovar 5, which is fundamental for the study of advanced gene screening. Inaddition, primer design was performed based on haemophilus influenzae in this study. In addition, PCR was performed by using genomic DNA of haemophilus parasuisserovar 5 as the template. The results demonstrated that we obtained two neo-gene:23SrRNA gene(conserved gene belonging to the large-subunit of ribosome) and adenylatecyclase gene(encodes adenylate cyclase and participates in converting adenyl nucleosidetriphosphate to cyclic adenosine3",5"-monophosphate). Furthermore, the phylogeneticanalyses between the species was performed, and neighbor-joining tree was constructedbased on comparison of 23S rRNA gene sequences, so it was illuminated betweenHaemophilus parasuis and other species in molecular evolution relationship.

选择我国流行优势菌株副猪嗜血杆菌血清5型地方株为研究对象，提取细菌基因组DNA，用限制性内切酶Sau3AⅠ对基因组DNA进行部分酶切，回收大小为1～5Kb的DNA片段，将其连接入原核表达载体pET-28a-c，最后转化宿主菌，结果成功地构建了基因表达文库，为后续的基因筛选工作奠定基础；另外，本研究选择嗜血杆菌属的流感嗜血杆菌为参考对象进行引物的设计，以副猪嗜血杆菌血清5型地方菌株的基因组DNA为模板，进行PCR扩增反应，结果表明成功地获得两个新基因：23S rRNA基因(存在于核糖体大亚基中的保守性基因)和腺苷酸环化酶基因(负责将腺嘌呤核苷三磷酸转变为环腺苷酸)，并进一步做了不同物种之间的分子系统发育分析，构建了基于23S rRNA基因的邻接法系统发育树，阐明了副猪嗜血杆菌与其它菌种的分子进化关系。

To date, only the structures of prokaryotic succinate:ubiquinone oxidoreductases, which share a similar enzymatic function with mitochondrial SQR, have been reported, including QFR from E.coli, QFR from Wolinella succinogenes and SQR from E.coli.

在此之前，只有原核生物的琥珀酸泛醌氧化还原酶的结构得到了解析，这包括大肠杆菌的 QFR ， Wolinella succinogenes 细菌中的 QFR 和大肠杆菌的 SQR ，这些蛋白有着与线粒体复合物 II 相类似的酶功能。

The homeodomain is responsible for binding to DNA, and experiments to swap homeodomains between proteins suggest that the specificity of DNA recognition lies within the homeodomain, but (like the situation with phage repressors) no simple code relating protein and DNA sequences can be deduced.

同源域负责结合DNA，在蛋白质之间交换同源域的实验证实了这一结论。但（就像噬菌体阻遏子的情形一样）并没有推出联系蛋白质与DNA序列的简单机理。同源域的C端和原核生物阻遏子α-β-α超二级结构有一定的同源关系。我们回想第13章，λ阻遏子有一个&识别螺旋&（α-螺旋-3）DNA的大构接触，而另一个螺旋（α-螺旋-2）位于 DNA的拐角。

Oocyte samples from one group were collected to detect the presence and integration of HBV DNA within cells and chromosomes using PCR, Southern blot, dot hybridization and fluorescence in situ hybridization. The female animals from another group were mated with their normal males, respectively. Their zygotes, 2-cell embryos were collected to detect the integration of HBV DNA in the female pronuclei of zygotes and the replication and expression of HBV genes in the 2-cell embryos using FISH, RT-PCR and immunofluoresence assay.(1) PCR detected positive bands in the tested oocyte samples fromgoldon hamster and mice. Southern blot revealed clear hybridization signals in PCR products.

研究用金黄地鼠和小鼠建立实验动物模型：将卵巢内注射HBV DNA的实验动物分成两组，一组注射后进行超排卵，收集卵巢和输卵管的卵母细胞，用PCR、Southern杂交，斑点杂交和荧光原位杂交(fluorescence in situ hybridization、FISH)检测HBV在卵母细胞内的存在和染色体上的整合；另一组超排雌鼠与正常雄鼠合笼，收集受精卵和2-细胞胚，用FISH、RT-PCR和免疫荧光检测技术分别研究HBV基因在受精卵雌原核上的整合以及在2-细胞胚中的复制与表达。

Under the same conditions pET28a-△SIL-2 expression was higher. RNA structure software analysis showed that mRNA5'end sequences of pET28a-IL-2 formed a complex and relatively stable secondary structure, which testified that the existence of the signal peptide sequence had influence on the ChIL-2 gene in prokaryon expression.

RNAstructure软件分析表明，pET28a-IL-2的mRNA5'端序列形成了复杂且较为稳定的二级结构，不利于翻译的起始，证明信号肽序列的存在对ChIL-2基因在原核细胞中的表达有一定的影响。

The apxⅢA gene of Actinobacillus pleuropneumonie was amplified by PCR. The amplified DNA fragment 3 466bp was cloned into pMD18-T. After R.E. analysis and sequencing, the apxⅢA gene in pMD18-T was ligated into pBluescripⅡSK, the recombinant expression plasmid pET-28b/apxⅢA was constructed and analysed with R.E., the protein of apxⅢA gene expressed in E.coli BL21 was detected by Western blotting.

因胸膜肺炎放线杆菌的致病性主要是由毒素决定的，故参照猪胸膜肺炎放线杆血清2型菌株的序列(GenBankL1 2 1 4 5)设计了一对特异性引物，用PCR的方法扩增apxⅢA基因并得到了长 3 466bp的片段，然后将其克隆到pMD 1 8T中，经酶切鉴定和序列分析表明克隆是成功的；再将apxⅢA插入到原核表达载体pET 2 8b后，转化BL2 1 (DE3)，在IPTG诱导下获得高效表达，经Westernblotting检测证实表达产物有活性。

I didn't watch TV last night, because it .

昨晚我没有看电视，因为电视机坏了。

Since this year, in a lot of villages of Beijing, TV of elevator liquid crystal was removed.

今年以来，在北京的很多小区里，电梯液晶电视被撤了下来。